Inhibition of AKT enhances mitotic cell apoptosis induced by arsenic trioxide

Toxicology and Applied Pharmacology 267 (2013) 228–237

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Inhibition of AKT enhances mitotic cell apoptosis induced by arsenic trioxide Ling-Huei Yih ⁎, Nai-Chi Hsu, Yi-Chen Wu, Wen-Yen Yen, Hsiao-Hui Kuo Institute of Cellular and Organismic Biology, Academia Sinica, Taipei 115, Taiwan, ROC

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Article history: Received 30 August 2012 Revised 6 January 2013 Accepted 10 January 2013 Available online 23 January 2013 Keywords: Arsenic trioxide AKT Mitotic arrest Spindle abnormalities Apoptosis

a b s t r a c t Accumulated evidence has revealed a tight link between arsenic trioxide (ATO)-induced apoptosis and mitotic arrest in cancer cells. AKT, a serine/threonine kinase frequently over-activated in diverse tumors, plays critical roles in stimulating cell cycle progression, abrogating cell cycle checkpoints, suppressing apoptosis, and regulating mitotic spindle assembly. Inhibition of AKT may therefore enhance ATO cytotoxicity and thus its clinical utility. We show that AKT was activated by ATO in HeLa-S3 cells. Inhibition of AKT by inhibitors of the phosphatidyl inositol 3-kinase/AKT pathway significantly enhanced cell sensitivity to ATO by elevating mitotic cell apoptosis. Ectopic expression of the constitutively active AKT1 had no effect on ATO-induced spindle abnormalities but reduced kinetochore localization of BUBR1 and MAD2 and accelerated mitosis exit, prevented mitotic cell apoptosis, and enhanced the formation of micro- or multi-nuclei in ATO-treated cells. These results indicate that AKT1 activation may prevent apoptosis of ATO-arrested mitotic cells by attenuating the function of the spindle checkpoint and therefore allowing the formation of micro- or multi-nuclei in surviving daughter cells. In addition, AKT1 activation upregulated the expression of aurora kinase B (AURKB) and survivin, and depletion of AURKB or survivin reversed the resistance of AKT1-activated cells to ATO-induced apoptosis. Thus, AKT1 activation suppresses ATO-induced mitotic cell apoptosis, despite the presence of numerous spindle abnormalities, probably by upregulating AURKB and survivin and attenuating spindle checkpoint function. Inhibition of AKT therefore effectively sensitizes cancer cells to ATO by enhancing mitotic cell apoptosis. © 2013 Elsevier Inc. All rights reserved.

Introduction Arsenic trioxide (ATO) alone induces remission in both primary and relapsed acute promyelocytic leukemia (APL) patients carrying the promyelocytic leukemia (PML)/retinoic acid receptor α (RARA) fusion oncoprotein (Chen et al., 2001; Soignet et al., 1998). However, ATO has not been successful in treating other types of cancers. In addition, chronic exposure to inorganic arsenic is carcinogenic (IARC, 2004; Straif et al., 2009). ATO prolongs the cardiac QT interval leading to torsade de pointes (Barbey et al., 2003). The US FDA-approved formulation of ATO induces APL differentiation syndrome, neuropathy, hepatotoxicity, and hematologic toxicity in patients with a variety of hematologic and solid malignancies (Douer and Tallman, 2005). Hence, approaches to reduce ATO toxicity and/or improve its clinical outcomes are essential. The induction of apoptosis and partial differentiation has been found to be the mechanism of action of ATO in APL (Douer and Tallman, 2005; Miller et al., 2002). Although ATO-induced APL cell apoptosis is associated with PML-RARA degradation (Shao et al., 1998), ATO also induces apoptosis in other malignant cells that lack this oncogenic fusion protein (Evens et al., 2004; Gazitt and Akay, 2005), indicating ATO may have other targets in different cellular

⁎ Corresponding author. Fax: +886 2 27858059. E-mail address: [email protected] (L.-H. Yih). 0041-008X/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.taap.2013.01.011

contexts. Because of the potential multiplicity of targets and complex mechanisms of action, ATO is broadly tested in combination with other agents in a variety of tumor cells (Dilda and Hogg, 2007; Litzow, 2008). Accumulated studies have demonstrated that ATO induces disorganized mitotic spindles and abnormal chromosome segregation and consequently results in mitotic arrest and apoptosis in malignant cells lacking functional p53 (Cai et al., 2003; Huang and Lee, 1998; Li and Broome, 1999; Ling et al., 2002; McNeely et al., 2006; Taylor et al., 2006; Yih et al., 2005). ATO-induced mitotic cell apoptosis may thus underlie its therapeutic effect. How arsenite disrupts mitotic spindles remains unclear. Arsenite alters in vitro tubulin polymerization, but there is disagreement among the reported results (Huang and Lee, 1998; Li and Broome, 1999; Ling et al., 2002), indicating other factors might be involved in the anti-mitotic effects of arsenite (Taylor et al., 2008). In addition to direct disruption of microtubules, disruption of proteins involved in regulating mitotic spindle assembly also induces defects of mitotic spindles and mitotic cell death (Janssen and Medema, 2011). Combination treatments that induce multiple disruptions of mitotic spindles and the regulatory machinery may improve the anti-tumor effects of individual drugs (Lee et al., 2007; Marcus et al., 2005). Therefore, combining ATO with other mitosis-disrupting agents might enhance its anti-tumor effect, effectively manage its toxic effects, and broaden its clinical utility. Functional activation of the spindle checkpoint resulting in mitotic arrest is required for apoptosis induction in response to microtubule-

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disrupting agents (Masuda et al., 2003; Sudo et al., 2004; Vogel et al., 2005). However, partial dysfunction of the spindle checkpoint occurring through altered transcriptional regulation of spindle checkpoint proteins by tumor suppressors or oncogene products is a frequent event in human tumors (Kops et al., 2005) and has been reported to result in premature exit from mitosis, generation of daughter cells with micro- or multi-nuclei, and a significant decrease in sensitivity of tumor cells to microtubule-disrupting drugs (Masuda et al., 2003; Sudo et al., 2004; Swanton et al., 2007; Tao et al., 2005; Yamada and Gorbsky, 2006). Because arsenite-induced mitotic cell apoptosis may contribute to its therapeutic effect (Cai et al., 2003; Gazitt and Akay, 2005), cancer cell response to arsenite-induced mitotic damage might be modulated by disruption of spindle checkpoint function. The serine-threonine kinase AKT, frequently over-activated in diverse tumors, transmits signals that regulate metabolism, cell cycle progression, and cell survival. Once activated by phosphorylation, AKT phosphorylates forkhead transcription factors, glycogen synthase kinase 3 (GSK3), BAD, and MDM2, resulting in antiapoptotic and/or cell survival signaling and consequently inducing drug resistance (McCubrey et al., 2011; Woodgett, 2005). AKT also stimulates DNA synthesis and cell cycle progression (Chang et al., 2003; Liang and Slingerland, 2003). Its constitutive activation can suppress DNA damage processing and lead to defects in DNA damage checkpoint control (Xu et al., 2010). AKT also promotes mitotic entry (Roberts et al., 2002) and induces polyploidization (Hixon et al., 2000). Furthermore, AKT regulates centrosome migration and spindle orientation in the early Drosophila melanogaster embryo (Buttrick et al., 2008). Inhibition of AKT interferes with centrosome function, induces spindle abnormalities, retards mitotic progression (Liu et al., 2008), and induces mitotic catastrophe in cancer cells (Hemstrom et al., 2006). Thus, AKT not only controls cell proliferation and survival but also regulates mitotic progression and assembly of mitotic spindles. A recent genomic approach demonstrated an association between AKT activation and resistance to Taxol, a microtubule stabilizing agent, in cancer cells (Riedel et al., 2008). We have previously demonstrated that ATO-induced mitotic cell apoptosis depends on a functional spindle checkpoint and that cancer cells with attenuated spindle checkpoint function were more resistant to ATO (Wu et al., 2008). Because AKT regulates the assembly of functional mitotic spindles and may lead to cell cycle deregulation and defects in checkpoint control in human cancers, we investigated whether and how AKT might modulate cell responses to ATO-induced spindle abnormalities and mitotic cell apoptosis.

Materials and methods Cell culture. HeLa-S3 cells were obtained from the American Type Culture Collection (Manassas, VA). The CGL-2 cell line (Stanbridge et al., 1981), derived from a hybrid (ESH5) of the HeLa variant D98/AH2, and a normal human fibroblast strain GM77, was kindly provided by Dr. E. J. Stanbridge (University of California-Irvine). The cells were maintained in Dulbecco's modified Eagle medium (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (Invitrogen), 0.37% sodium bicarbonate, 100 U/ml penicillin, and 100 μg/ml streptomycin at 37 °C in an humidified incubator in air containing 10% CO2 and were passaged twice per week. Stable cell clones (CGL2-X and Myr-AKT1) were generated by transfecting CGL-2 cells with pUSE (a cytomegalovirus-neo empty vector control) or MYC-tagged expression vector pUSE-Myr-AKT1 (a constitutively active and membrane-targeting myristoylated AKT1 construct (Kohn et al., 1996)) and selecting with 0.5 mg/ml G418 sulfate (Invitrogen) for 2 weeks. Cells were synchronized at G1 by the double-thymidine block protocol and enriched for S phase by release from thymidine block for 3 h (Yih et al., 2006).

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Drug treatment. Logarithmically growing or synchronized cells were left untreated, were treated with 1–4 μM ATO or each inhibitor alone, or were co-treated with ATO plus each inhibitor for various periods. They were then harvested and examined for growth inhibition, cell cycle distribution, or apoptosis, or by immunofluorescence staining or immunoblotting. A stock solution of ATO (10 mM, Sigma, St. Louis, MO) was freshly prepared in 0.1 N NaOH and diluted in culture medium before use. Stock solutions (10 mM) of AKT inhibitor-VIII, LY294002, and potassium bisperoxo(bipyridine) oxovanadate (BpV) (Calbiochem, San Diego, CA) were prepared in dimethyl sulfoxide and stored at −20 °C. Analysis of cell cycle distribution. DNA was stained with propidium iodide and mitotic cells quantified by measuring the expression of the mitosis-specific marker, phospho-histone H3 (phospho-H3), as described (Yih et al., 2006). Phospho-H3 levels and the DNA content of individual cells were analyzed using a flow cytometer (FACSCanto II, BD Biosciences, San Jose, CA). Detection of apoptosis. The number of apoptotic cells was determined using an annexin V–fluorescein isothiocyanate (FITC) apoptosis detection kit (Calbiochem) as described (Yih et al., 2005). FITC binding was analyzed using the FACSCanto II flow cytometer (BD Biosciences), and the percentage of apoptotic cells (FITC-positive) in 10,000 cells was calculated. Apoptosis was also determined by flow cytometry analysis of cleaved poly(ADP-ribose) polymerase (PARP) in individual cells relative to DNA content (Li and Darzynkiewicz, 2000). After drug treatment, cells were fixed with ice-cold 70% ethanol for 16 h and then simultaneously immunostained for 3 h with a rabbit antibody against cleaved PARP (#9541, Cell Signaling Technology, Danvers, MA) and mouse anti-phospho-H3 (serine 10) (#9706, Cell Signaling Technology), followed by incubation for 1 h with FITC-conjugated goat anti-mouse IgG and allophycocyanin-conjugated goat anti-rabbit IgG (Invitrogen). The cells were then stained with 4 μg/ml of propidium iodide in phosphate-buffered saline (PBS, pH 7.4) containing 1% Triton X-100 and 0.1 mg/ml RNase A. The level of cleaved PARP, phospho-H3, and DNA content of individual cells were analyzed using a flow cytometer (FACSCanto II, BD Biosciences). Growth inhibition and colony formation assays. Cell growth was determined by assaying viable cell numbers using methylthiazole tetrazolium (WST-8) (Cell Count kit 8; Dojindo Molecular Technologies, Inc., Gaithersburg, MD) as described (Wu et al., 2009). Cells were seeded in a 96-well plate (3000/well) and after 24 h were treated with ATO and/or inhibitors for 72 h. WST-8 was added to the medium at the end of treatment and the plates incubated at 37 °C for 1 h, then cell viability was determined by optical absorption (450 nm) of the reduced formazan. Cell growth was determined from the absorption at 450 nm and expressed as a percentage of that of control cultures. For the colony formation assay, the treated cells were collected, counted, serially diluted, and seeded in triplicate at a density of 200 to 2000 cells/dish in 60-mm Petri dishes and incubated for 10 days. Colonies were visualized and counted after fixing with 70% ethanol and staining with 3% Giemsa solution (Merck, Darmstadt, Germany). The plating efficiency of each treatment was calculated by dividing the numbder of colonies on each plate by the number of cells seeded. Immunofluorescence staining. Cells seeded on glass coverslips were treated with ATO and/or inhibitors for 24 h at 37 °C, then were washed twice with PBS and fixed in situ with 90% methanol at − 20 °C for 10 min. The cells were again washed twice with PBS and immunostained for 1 h at room temperature with anti-αtubulin (clone B512, Sigma), anti-BUBR1 (Chemicon, Temecula, CA), anti-MAD2 (Santa Cruz Biotechnology, Santa Cruz, CA), or anticentromere (Antibodies Incorporated, Davis, CA). The cells were

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processed as described (Yih et al., 2012) and examined under a fluorescence microscope (Axioskop 2, Zeiss, Oberkochen, Germany). To calculate the percentage of mitotic cells with abnormal mitotic spindles or the percentage of cells with micro- or multi-nuclei, three or four independent experiments were performed and at least 300 cells were analyzed in each experiment. Localization of BUBR1 or MAD2 at kinetochores in arrested mitotic cells was demonstrated by double staining cells with the anti-centromere antibody and anti-BUBR1 or anti-MAD2. The percentage of mitotic cells with positive BUBR1 or MAD2 staining in kinetochores was determined from at least 300 randomly selected mitotic cells in three independent experiments. Immunoblotting. Cell lysates were prepared, and equal amounts of cellular protein (10–30 μg) were resolved with SDS-PAGE, transferred to polyvinylidene difluoride membranes, and specific proteins were detected with immunoblotting as described (Yih et al., 2005). β-actin was used as the loading control. The results shown are representative of at least two independent experiments. The goat anti-AKT1 and rabbit anti-survivin were from Santa Cruz Biotechnology, the rabbit anti-phospho-AKT1 (S473 or T308), rabbit anti-aurora kinase B (AURKB), rabbit anti-phospho-glycogen synthetase kinase3β (pGSK3β-S9), and rabbit anti-phospho-S6K (pS6K-T389) from Cell Signaling Technology, and the mouse anti-β-actin (MAB1501) from Chemicon. The monoclonal anti-MYC (9E10) was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by The University of Iowa, Department of Biological Sciences (Iowa City, IA). RNA interference. The small interfering RNAs (siRNAs) (On-Target plus SMART pool) were chemically synthesized by Thermo Scientific Dharmacon (Lafayette, CO). Cells were plated at a density of 0.5 to 1 × 10 5 per 35-mm dish one day before transfection, then were transfected with 50 nM AURKB or survivin siRNA using Oligofectamine (Invitrogen). At 24 h after transfection, the medium was replaced with fresh medium and the cells treated with ATO for 24 or 48 h. A double-stranded RNA targeting luciferase (5′cguacgcggaauacuucgadTdT-3′) was used as the control. Statistics. Data are presented as the mean ± standard deviation (SD) of 3–4 independent experiments. Statistical analysis was performed with PRISM v5.0 (GraphPad Inc., San Diego, CA) using the Student's t-test. p b 0.05 was considered statistically significant. Results AKT1 is activated by ATO and Protects Cells from ATO Cytotoxicity The effects of ATO on AKT activation are shown in Fig. 1A. ATO treatment for 8 h induced AKT1 phosphorylation at serine 473 (pAKT1 S473) and threonine 308 (pAKT1 T308), and a concomitant increase in the expression of phosphorylated GSK3β (pGSK3β S9) and S6K (pS6K T389), two proteins that are phosphorylated after activation of AKT, in HeLa-S3 cells, indicating that ATO may activate AKT1. To evaluate the role of AKT in ATO cytotoxicity, HeLa-S3 cells were treated with ATO alone or ATO plus LY294002 (a general inhibitor of phosphatidyl inositol 3-kinase (PI3K)/AKT pathway), AKT inhibitor-VIII (an AKT specific inhibitor), or BpV (a phosphatase inhibitor that has been reported to inhibit PTEN (Pi et al., 2012)). Treatment of HeLa-S3 cells with ATO resulted in a dose-dependent induction of mitotic arrest at 24 h (Fig. 1B) and apoptosis at 72 h (Fig. 1C) and a consequent decrease in cell viability (Fig. 1D). Treatment with ATO plus LY294002 or AKT inhibitor-VIII significantly enhanced ATOinduced mitotic arrest and apoptosis and reduced cell viability (Figs. 1B–D). In contrast, treatment with ATO plus BpV significantly reduced ATO-induced mitotic arrest, apoptosis, and cell death (Figs.

1B–D). Thus, the AKT pathway might protect cancer cells from ATO-induced mitotic arrest and apoptosis. LY294002 enhances ATO cytotoxicity by promoting mitotic cell apoptosis We then examined how LY294002 enhances ATO cytotoxicity. Consistent with a previous study (Wu et al., 2009), treatment of HeLa-S3 cells with 2 μM ATO for 24 h induced abnormalities in mitotic spindles (Fig. 2A). As Fig. 2B shows, 25% of the mitotic cells arrested by ATO contained abnormal mitotic spindles. LY294002 co-treatment significantly increased the percentage of abnormal mitotic cells to 47% (Fig. 2B). In addition, 2 μM ATO induced a small increase in mitotic cells at 24 h. These cells then resumed cell cycle progression with little induction of apoptosis (Fig. 2C). LY294002 greatly enhanced ATO-induced mitotic cell accumulation at the expense of the G1 fraction from 24 h onward, with no significant changes in the number of S or G2 cells. After 36 h, the percentage of mitotic cells decreased and the percentages of sub-G1 and annexin V– positive apoptotic cells showed a large increase (Fig. 2C). These results indicated that LY294002 might sensitize HeLa-S3 cells to ATO by causing an increase in mitotic abnormalities, mitotic arrest, and mitotic cell apoptosis. To confirm this, HeLa-S3 cells were then treated for 24 h with 2 μM ATO alone or in combination with LY294002, then the floating mitotic cells were shaken off, re-incubated for different times with the same agent (ATO alone or ATO plus LY294002), and their cell cycle stage and PARP cleavage examined. The mitotic cells from cultures treated with 2 μM ATO alone exited mitosis and entered G1 at 6 h of re-incubation, S phase at 10–16 h, and G2/M stage at 16 h (Fig. 3A), confirming that these mitotic cells resumed cell cycle progression. However, the mitotic cells from cultures co-treated with 2 μM ATO and LY294002 mainly underwent apoptosis, as reflected by a dramatic increase in cells showing PARP cleavage and only a slight increase in the numbers of G1 and S cells during re-incubation (Fig. 3B). These results confirmed that LY294002 sensitizes HeLa-S3 cells to ATO by enhancing mitotic cell apoptosis. AKT1 activation disrupts spindle checkpoint function, prevents mitotic cell apoptosis, and enhances the formation of micro- or multi-nuclei in ATO-treated cells Because AKT was activated by ATO and inhibition of AKT enhanced ATO-induced mitotic cell apoptosis, we then examined whether AKT activation could prevent mitotic arrest and spindle abnormalities in ATO-treated cells. Because CGL2 cells are extremely sensitive to ATO-induced mitotic cell apoptosis (Wu et al., 2008) and AKT1 is the most ubiquitous type of AKT (Luo et al., 2003), stable CGL2 cell clones were established expressing the constitutively active and membrane-targeting myristoylated AKT1 (Myr-AKT1) and the empty vector control (CGL2-X). Similar to what was found in HeLa-S3 cells, ATO induced AKT phosphorylation at S473 in CGL2-X cells (Fig. 4A). The expression of the MYC-tagged AKT1 mutant was evident in the Myr-AKT1 cells (Fig. 4A). AKT1 phosphorylation was considerably higher in untreated Myr-AKT1 cells and remained high after ATO treatment (Fig. 4A). GSK3β was also highly phosphorylated in the Myr-AKT1 cells compared to the CGL2-X cells in the presence or absence of ATO (Fig. 4A), confirming that AKT was constitutively activated in the Myr-AKT1 cells. The role of AKT1 activation in ATO-induced mitotic defects was then examined. ATO-induced spindle abnormalities were not affected by overexpression of Myr-AKT1 (Fig. 4B), but ATO-induced mitotic arrest (Fig. 4C) and apoptosis (Fig. 4D) were greatly reduced in the Myr-AKT1 cells compared to the CGL2-X cells. To explore how Myr-AKT1 overexpression prevents ATO-induced mitotic arrest and apoptosis, CGL2-X and Myr-AKT1 cells were synchronized at G1 by double thymidine block. At 3 h after release from block, the cells were synchronized in S phase and were treated with

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Fig. 1. AKT1 is activated by ATO and protects cells from ATO cytotoxicity. (A) ATO-induced AKT1 activation. HeLa-S3 cells were treated for 8 h with 0–5 μM ATO, then expression of AKT1, phosphorylated AKT1 (pAKT1S473 and pAKT1T308), phosphorylated GSK3β (pGSK3βS9), and phosphorylated S6K (pS6KT389) were analyzed by immunoblotting. (B–D) Inhibition of AKT enhances ATO-induced mitotic arrest, apoptosis, and cell death. HeLa-S3 cells were treated with 0–4 μM ATO alone or in combination with 5 μM AKT inhibitor-VIII, 5 μM LY294002, or 10 μM BpV and were analyzed for cell cycle stage at 24 h by flow cytometry of phospho-H3 and DNA content (B), for apoptosis at 72 h by flow cytometry of annexin V binding (C), and for cell viability at 72 h by WST-8 assay (D). * indicates p b 0.05 comparing the combined treatments with ATO alone (Student's t test).

1 μM ATO. Flow cytometric analysis showed that untreated CGL2-X cells progressed into G2 at 6 h and mitosis at 10 h after thymidine release and had exited from mitosis and entered G1 by 12 h (Fig. 4E, upper panel). Most of the ATO-treated CGL2-X cells also progressed into G2 at 6 h and entered mitosis at 10 h but then arrested and underwent apoptosis, as reflected by a dramatic increase in cleaved PARP–positive cells at 18 h (Fig. 4E, upper panel). Untreated MyrAKT1 cells progressed through S phase and entered G2 and mitosis 2 h earlier than CGL2-X cells (Fig. 4E, lower panel), indicating that expression of Myr-AKT1 accelerated S phase progression and promoted the G2/M transition. ATO-treated Myr-AKT1 cells progressed into G2 and M phase in the same way as untreated Myr-AKT1 cells, then divided at 12 h after thymidine release, as revealed by the considerable increase in G1 cells with no significant induction of apoptosis (Fig. 4E, lower panel). These results indicated that ATO-arrested mitotic Myr-AKT1 cells exited from mitosis faster than the CGL2-X cells and resumed cell cycle progression with little apoptosis. Because the spindle checkpoint is the major control mechanism for mitotic arrest and is required for arsenite-induced mitotic cell apoptosis (McNeely et al., 2008a, 2008b; Wu et al., 2008), its function in CGL2-X and Myr-AKT1 cells was assessed by the kinetochore localization of BUBR1 and MAD2 (Musacchio and Hardwick, 2002). In

untreated CGL2-X or Myr-AKT1 cells, BUBR1 and MAD2 signals were detectable at kinetochores in almost all the metaphase-like cells, indicating that these two cell clones had functional spindle checkpoint and that overexpression of activated AKT1 had no significant effect on BUBR1 and MAD2 localization in unstressed condition. BUBR1 and MAD2 signals were also visible at kinetochores in > 90% of ATO-arrested mitotic CGL2-X cells (Fig. 5A). However, localization of BUBR1 and MAD2 to kinetochores was significantly reduced in ATO-arrested mitotic Myr-AKT1 cells (Fig. 5A), indicating that spindle checkpoint function might be compromised in these cells. In addition, formation of micro- or multi-nuclei was only slightly induced by ATO in CGL2-X cells but was considerably increased in Myr-AKT1 cells (Fig. 5B). The colony-forming ability of ATO-arrested mitotic Myr-AKT1 cells was also significantly higher than that of ATOarrested CGL2-X cells (Fig. 5C), indicating that the arrested mitotic Myr-AKT1 cells could escape apoptosis and resume cell proliferation. Collectively, these results indicated that AKT1 activation might disrupt the activation of spindle checkpoint proteins, hence reducing mitotic cell accumulation, preventing mitotic cell apoptosis, and allowing the formation of micro- or multi-nuclei in daughter cells and the proliferation of surviving cells despite the spindle abnormalities frequently present in ATO-arrested mitotic Myr-AKT1 cells.

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ATO ( M, 24 h) Fig. 2. LY294002 enhances ATO-induced mitotic defects. (A) Representative immunofluorescence images of normal and ATO-arrested mitotic cells. HeLa-S3 cells were left untreated or treated with 2 μM ATO for 24 h and then fixed and immunostained with anti-α-tubulin to detect mitotic spindles (green) and counterstained with DAPI to detect chromosomes (blue). Scale bar, 10 μm. (B) LY294002 increases ATO-induced spindle abnormalities. HeLa-S3 cells were treated with 2 μM ATO alone or in combination with 5 μM LY294002 and analyzed for mitotic spindles at 24 h by immunofluorescence staining as described in (A). The results shown represent the mean ± SD of 300 mitotic cells from three independent experiments. ** indicates p b 0.01 compared to ATO alone (Student's t test). (C) LY294002 enhances ATO-induced mitotic arrest and apoptosis in HeLa-S3 cells. HeLa-S3 cells were treated as in (B) and analyzed for cell cycle stage and apoptosis at 24–72 h by flow cytometry of phospho-H3, DNA content, and annexin V binding.

division (Li et al., 1998; Terada et al., 1998). ATO induced significantly higher apoptosis in CGL2-X cells than in Myr-AKT1 cells (Fig. 6C), confirming that overexpression of Myr-AKT1 could protect cells from ATO-induced mitotic cell apoptosis. siRNA-mediated depletion of AURKB or survivin was confirmed by immunoblotting (Figs. 6A and B) and, in the absence of ATO, induced significantly more apoptosis in CGL2-X cells than in Myr-AKT1 cells (Fig. 6C), indicating that AKT1 activation might protect cells from defects induced by depletion of AURKB or survivin. Depletion of either of these two proteins did not further increase ATO-induced apoptosis in CGL2-X cells but greatly sensitized Myr-AKT1 cells to ATO-induced apoptosis to a similar extent as that in ATO-treated CGL2-X cells (Fig. 6C). In addition, the colony-forming ability of ATO-arrested mitotic Myr-AKT1

Downregulation of AURKB or Survivin Sensitizes Myr-AKT1 Cells to ATOinduced Apoptosis Upregulation of survivin and/or aurora kinases has been reported to be responsible for AKT-mediated resistance to microtubuledisrupting agents (Lu et al., 2009; Wang et al., 2006). In the absence of ATO, expression of AURKB (Fig. 6A) and survivin (Fig. 6B) in Myr-AKT1 cells was higher than that in CGL2-X cells, indicating that AKT1 activation might upregulate the expression of AURKB and survivin. Levels of AURKB and survivin were significantly increased by ATO in CGL2-X cells but not in Myr-AKT1 cells, suggesting induction of severe mitotic arrest in CGL2-X cells, as expression of AURKB and survivin peak during G2 and mitosis and decrease after cell

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Fig. 3. LY294002 enhances ATO cytotoxicity by promoting mitotic cell apoptosis. HeLa-S3 cells were treated for 24 h with 2 μM ATO alone or in combination with 5 μM LY294002, then the floating mitotic cells were collected and re-incubated for 0–24 h in medium containing ATO (A) or ATO plus LY294002 (B), and cell cycle stage and PARP cleavage were analyzed by flow cytometry.

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Time after thymidine release (h) Fig. 4. AKT1 activation reduces ATO-induced mitotic arrest and mitotic cell apoptosis but does not affect the induction of spindle abnormalities. (A) Immunoblot analysis of stable clones transfected with empty vector (CGL2-X) or a MYC-tagged constitutively active AKT1 (Myr-AKT1). The cells were treated with the indicated concentration of ATO for 8 h, and cell lysates were prepared for immunoblotting. The black arrowhead indicates endogenous AKT1, and the gray arrowhead indicates ectopically expressed AKT1. (B–D) Effects of overexpression of the constitutively active AKT1 on ATO-induced spindle abnormalities (B), mitotic arrest (C), and apoptosis (D). CGL2-X or Myr-AKT1 cells were left untreated or were treated with 1.5 μM ATO and analyzed for mitotic spindles at 24 h by immunofluorescence staining of α-tubulin, for cell cycle stage at 24 h by flow cytometry of phospho-H3 and DNA content, and for apoptosis at 48 h by flow cytometry of PARP cleavage. # indicates pb 0.05 (Student's t test) compared to untreated CGL2-X cells. ** indicates p b 0.01 (Student's t test) compared to ATO-treated CGL2-X cells. (E) Overexpression of the constitutively active AKT1 facilitates mitosis exit and prevents ATO-induced mitotic cell apoptosis. CGL2-X cells (upper) or Myr-AKT1 cells (lower) were enriched at S phase, then were left untreated or were treated with 1 μM ATO for the indicated time when the cells were analyzed for cell cycle stage and PARP cleavage.

cells was also significantly reduced by siRNA-mediated depletion of AURKB or survivin (Fig. 5C). These results suggest that the severe damages induced by ATO in aberrant mitotic CLG2-X cells might not be ameliorated by endogenous AKT and hence the resulting high level of apoptosis could not be further enhanced by depletion of AURKB or survivin. Alternatively, ATO-induced mitotic damage might alter the signaling pathway upstream of AURKB and survivin, therefore depletion of AURKB or survivin did not further enhance ATO-induced mitotic cell apoptosis. Furthermore, the resistance to ATO-induced mitotic cell apoptosis in Myr-AKT1 cells can be reversed by depletion of AURKB or survivin, indicating that AKT1 activation might suppress ATO-induced mitotic cell apoptosis, at least in part, by upregulation of AURKB and survivin. Discussion Our results suggest combination with AKT while minimizing ATO has been reported to

that, in cancer treatment, the use of ATO in inhibitors may enhance therapeutic efficacy dose and thus its toxic side effects. Arsenite promote the proliferation of keratinocytes

through AKT-mediated cyclin D accumulation (Ouyang et al., 2007) and to induce migration and invasion of bronchial epithelial cells through AKT-mediated expression of zinc-finger E-box-binding homeobox factors (Wang et al., 2012). The arsenite-induced AKT activation has been demonstrated to be associated with downregulation of protein phosphatase 2A and pleckstrin homology domain leucine-rich repeat protein phosphatase 2 (Zhang et al., 2011), phosphatases known to induce AKT dephosphorylation and inactivation (Trotman et al., 2006). Arsenite also was reported to induce AKT activation through JNK-dependent phosphorylation of signal transducer and activator of transcription 3 (Liu et al., 2012). In contrast, arsenite has been reported to induce B-cell chronic lymphocytic leukemia cell apoptosis through inactivation of AKT due to upregulation of PTEN (Redondo-Munoz et al., 2010). It has also been shown that arsenite inhibits AKT function and decreases AKT protein levels in a caspase-dependent manner (Mann et al., 2008), which would induce apoptosis (Choi et al., 2002). The arsentieinduced suppression of AKT signaling was also evident in embryonic and neural stem cells (Ivanov and Hei, in press; Ivanov et al., 2013). It is possible that arsenite at non-lethal concentrations might activate

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Myr-AKT1

Untreated

100

**

40 20 0

BUBR1

BUBR1

MAD2

No ATO

15 10

* 5 0

MAD2

*

&

#

#

# # & #

20

* *

10 #

0 1.5

CGL2-X

ATO 1.5 M

60

&

0

0 1.5 ATO ( M)

Myr-AKT1

CGL-X

si-control si-AURKB si-Survivin

**

20

80

si-control si-AURKB si-Survivin

60

**

si-control si-AURKB si-Survivin

80

**

25

ATO 1 M

100

Plating efficiency (%)

CGL2-X

% of cells with micronuclei or multinuclei

% of mitotic cells with BUBR1 or MAD2 located at kinetochores

C

B

A

si-control si-AURKB si-Survivin

234

Myr-AKT1

Fig. 5. AKT1 activation disrupts spindle checkpoint activation and enhances the formation of micro- or multi-nuclei in ATO-treated cells. (A) Overexpression of the constitutively active AKT1 reduces localization of BUBR1 and MAD2 at kinetochores. Cells were untreated or treated with 1.5 μM ATO for 24 h, and fixed. Localization of BUBR1 or MAD2 at kinetochores in mitotic cells was demonstrated by double staining cells with anti-centromere antibody and anti-BUBR1 or anti-Mad2. ** indicates p b 0.01 (Student's t test) compared to CGL2-X cells. (B) Overexpression of constitutively active AKT1 enhances ATO-induced micro- and multi-nuclei formation. CGL2-X or Myr-AKT1 cells were treated with 1.5 μM ATO for 24 h and analyzed for induction of micro- and multi-nuclei by immunofluorescence staining of nuclei with DAPI. (C) Overexpression of constitutively active AKT1 induces resistance to ATO. CGL2-X or. Myr-AKT1 cells were first transfected with control-, AURKB-, or survivin-specific siRNAs, then treated with 1 μM ATO for 24 h. The arrested mitotic cells were then collected and tested in the colony-forming assay. The data represent the relative plating efficiency compared to the untreated control in each stable clone. # indicates p b 0.05 (Student's t test) compared to untreated cells of each clone, & indicates p b 0.05 compared to CGL2-X cells with the same treatment, and * indicates p b 0.05 (Student's t test) compared to control siRNA–transfected Myr-AKT1 cells treated with 1.5 μM ATO.

B Myr-AKT1

0

0 1

1

Control

1

survivin

0

Control

0 1

Myr-AKT1

CGL2-X

AURKB

AURKB

0 1

Control

Control

CGL2-X

0 1

0 1

survivin

A

0

1 ATO ( M) survivin

AURKB 1.0 2.1 0.1 0.1

1.7 1.9 0.1 0.1

1.0 1.9 0.1 0.1

1.9 1.8 0.1 0.1

-actin

C

control siRNA

siRNA (50 nM)

-actin

survivin siRNA

AURKB siRNA

% of cleaved PARP+ cells

60

*

50

*

#

40 30 20

&

#

&

#

&

#

10 0

#

# 0

1.5

CGL2-X

0

1.5

Myr-AKT1

ATO ( M, 48 h)

Fig. 6. Depletion of AURKB and survivin sensitizes Myr-AKT1 cells to ATO-induced apoptosis. (A and B) AKT1 activation upregulates the expression of AURKB and survivin, and siRNA causes depletion of AURKB and survivin. CGL2-X or Myr-AKT1 cells were transfected with control-, AURKB-, or survivin-specific siRNAs, then, 24 h after transfection, were treated with 0 or 1 μM ATO and analyzed for expression of AURKB or survivin at 24 h by immunoblotting. The numbers below each blot indicate the ratio of the mean expression level of AURKB or survivin to that of the untreated CGL2-X cells transfected with control siRNA after normalization to the level of β-actin. (C) Depletion of AURKB or survivin sensitizes Myr-AKT1 cells to ATO. The cells were treated as in (A and B), but with 1.5 μM ATO, and analyzed for apoptosis at 48 h by flow cytometry of PARP cleavage. # indicates p b 0.05 (Student's t test) compared to untreated cells of each clone, & indicates p b 0.05 compared to CGL2-X cells with the same treatment, and * indicates p b 0.05 (Student's t test) compared to control siRNA–transfected Myr-AKT1 cells treated with 1.5 μM ATO.

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AKT, whereas at cytotoxic concentrations it might inhibit or limit AKT activation. Nonetheless, inhibition of the PI3K/AKT pathway prevents the mitogenic effects of arsenite (Wang et al., 2012) and/or further enhances its cytotoxicity (Redondo-Munoz et al., 2010) in diverse cell systems. Therapeutic inhibition of AKT may therefore greatly reduce the ATO concentration necessary to kill target cells. The activation of AKT, ERK, or p38 during treatment of malignant cells with ATO negatively controls cell response to ATO (Verma et al., 2002; Ramos et al., 2005; Lunghi et al., 2006). Our results showed that LY294002 or AKT inhibitor-VIII, but not PD98059 (an ERK inhibitor), SB-203580 (a p38 inhibitor), or SP600125 (a JNK inhibitor; data not shown), significantly enhanced ATO-induced mitotic arrest and apoptosis in HeLa-S3 cells. Previous studies revealed that LY294002 selectively augments the cytotoxicity of microtubule-disrupting agents (Riedel et al., 2008; Shingu et al., 2003), indicating that AKT might play a pivotal role in the response to spindle defects. AKT can be phosphorylated and activated by microtubule-disrupting agents, and its activation leads to some of the observed drug resistance of cancer cells (Liu et al., 2006; VanderWeele et al., 2004). It has also been reported that mitotic cell apoptosis can be initiated by dephosphorylation and activation of caspase-9 during prolonged mitotic arrest (Allan and Clarke, 2007). Because caspase-9 can be inactivated by AKT-mediated phosphorylation (Cardone et al., 1998), AKT activation might result in resistance to microtubule-disrupting agents through AKT-mediated phosphorylation and inactivation of caspase-9. However, our results showing that AKT inhibition enhances mitotic arrest whereas its activation decreases mitotic arrest in ATO-treated cells before initiation of apoptosis indicate that AKT might negatively control the induction of mitotic arrest. In addition, overexpression of constitutively active AKT1 did not affect ATO induction of mitotic abnormalities, but it did reduce mitotic cell accumulation by accelerating the exit of these abnormal cells from mitosis. Because ATO induced many mitotic abnormalities in both CGL2-X and Myr-AKT1 cells, AKT-mediated ATO resistance may not be due to the ability of AKT to inhibit ATO activity, either by directly inactivating ATO or by indirectly increasing ATO clearance from cells. In addition to directly preventing apoptosis by regulating the expression and activity of apoptotic proteins (Song et al., 2005), AKT activation also has been reported to promote DNA replication (Chang et al., 2003; Liang and Slingerland, 2003), promote cell cycle transition from S to G2 (Maddika et al., 2008) or from G2 to mitosis (Katayama et al., 2005), override the G2 DNA damage checkpoint (Xu et al., 2010), and regulate centrosome migration and spindle orientation (Buttrick et al., 2008). AKT also enhances cancer cell resistance to microtubule-disrupting agents through upregulation of survivin and/or aurora kinases (Lu et al., 2009; Wang et al., 2006). These observations suggest that AKT may promote cell survival through pathways other than direct regulation of apoptotic proteins. We demonstrated that AKT1 activation reduced the localization of BUBR1 and MAD2 at kinetochores in ATO-arrested mitotic cells, indicative of attenuated spindle checkpoint function (Musacchio and Hardwick, 2002). Chronic activation of AKT leads to multi-nuclei formation owing to a combination of endomitosis and cell fusion (Jin and Woodgett, 2005). AKT overexpression also leads to cell hypertrophy and polyploidization (Hixon et al., 2000). Because the spindle checkpoint is the major cell cycle control mechanism preventing chromosome missegregation and aneuploidy, these results imply that AKT activation may attenuate spindle checkpoint function. In addition, our results showed that AKT1 activation upregulated the expression of AURKB and survivin, proteins that have attracted considerable attention because of their roles in controlling mitosis fidelity (Lens et al., 2006; Vader and Lens, 2008) and their high overexpression in tumors (Altieri, 2003; Vischioni et al., 2006). Overexpression of AURKB and survivin in cancer cells correlates with resistance to microtubule-disrupting agents (Chieffi et al., 2006; Lu et al., 2009) and with the development of aneuploidy and/

235

or polyploidy (Connell et al., 2008; Ota et al., 2002), reflecting a defective spindle checkpoint in cells overexpressing these proteins. In addition, upregulation of survivin is mediated by PI3K/AKT and accelerates mitosis exit (Lu et al., 2009). Thus, AKT activation may alter the function of the spindle checkpoint by upregulating the expression of AURKB and/or survivin. We previously showed that ATO induces apoptosis in a spindle checkpoint–dependent manner (Wu et al., 2008; Yih et al., 2006). AKT activation may alter the function of the spindle checkpoint, facilitating mitotic exit and reducing mitotic arrest after ATO treatment despite the presence of spindle abnormalities and thus allowing micro- and multi-nuclei formation and proliferation of surviving cells. Inhibition of AKT therefore significantly enhances ATO-induced mitotic cell apoptosis. Funding This work was supported by Academia Sinica and a grant from National Science Council [NSC99-2320-B-001-008-MY3], Taiwan. Conflict of interest The authors have nothing to declare. Acknowledgments We thank the Core Facility of the Institute of Cellular and Organismic Biology, Academia Sinica, for assistance with flow cytometry and microscopy. References Allan, L.A., Clarke, P.R., 2007. Phosphorylation of caspase-9 by CDK1/cyclin B1 protects mitotic cells against apoptosis. Mol. Cell 26, 301–310. Altieri, D.C., 2003. Validating survivin as a cancer therapeutic target. Nat. Rev. Cancer 3, 46–54. Barbey, J.T., Pezzullo, J.C., Soignet, S.L., 2003. Effect of arsenic trioxide on QT interval in patients with advanced malignancies. J. Clin. Oncol. 21, 3609–3615. Buttrick, G.J., Beaumont, L.M., Leitch, J., Yau, C., Hughes, J.R., Wakefield, J.G., 2008. Akt regulates centrosome migration and spindle orientation in the early Drosophila melanogaster embryo. J. Cell Biol. 180, 537–548. Cai, X., Yu, Y., Huang, Y., Zhang, L., Jia, P.M., Zhao, Q., Chen, Z., Tong, J.H., Dai, W., Chen, G.Q., 2003. Arsenic trioxide-induced mitotic arrest and apoptosis in acute promyelocytic leukemia cells. Leukemia 17, 1333–1337. Cardone, M.H., Roy, N., Stennicke, H.R., Salvesen, G.S., Franke, T.F., Stanbridge, E., Frisch, S., Reed, J.C., 1998. Regulation of cell death protease caspase-9 by phosphorylation. Science 282, 1318–1321. Chang, F., Lee, J.T., Navolanic, P.M., Steelman, L.S., Shelton, J.G., Blalock, W.L., Franklin, R.A., McCubrey, J.A., 2003. Involvement of PI3K/Akt pathway in cell cycle progression, apoptosis, and neoplastic transformation: a target for cancer chemotherapy. Leukemia 17, 590–603. Chen, Z., Chen, G.Q., Shen, Z.X., Chen, S.J., Wang, Z.Y., 2001. Treatment of acute promyelocytic leukemia with arsenic compounds: in vitro and in vivo studies. Semin. Hematol. 38, 26–36. Chieffi, P., Cozzolino, L., Kisslinger, A., Libertini, S., Staibano, S., Mansueto, G., De Rosa, G., Villacci, A., Vitale, M., Linardopoulos, S., Portella, G., Tramontano, D., 2006. Aurora B expression directly correlates with prostate cancer malignancy and influence prostate cell proliferation. Prostate 66, 326–333. Choi, Y.J., Park, J.W., Suh, S.I., Mun, K.C., Bae, J.H., Song, D.K., Kim, S.P., Kwon, T.K., 2002. Arsenic trioxide-induced apoptosis in U937 cells involve generation of reactive oxygen species and inhibition of Akt. Int. J. Oncol. 21, 603–610. Connell, C.M., Wheatley, S.P., McNeish, I.A., 2008. Nuclear survivin abrogates multiple cell cycle checkpoints and enhances viral oncolysis. Cancer Res. 68, 7923–7931. Dilda, P.J., Hogg, P.J., 2007. Arsenical-based cancer drugs. Cancer Treat. Rev. 33, 542–564. Douer, D., Tallman, M.S., 2005. Arsenic trioxide: new clinical experience with an old medication in hematologic malignancies. J. Clin. Oncol. 23, 2396–2410. Evens, A.M., Tallman, M.S., Gartenhaus, R.B., 2004. The potential of arsenic trioxide in the treatment of malignant disease: past, present, and future. Leuk. Res. 28, 891–900. Gazitt, Y., Akay, C., 2005. Arsenic trioxide: an anti cancer missile with multiple warheads. Hematology 10, 205–213. Hemstrom, T.H., Sandstrom, M., Zhivotovsky, B., 2006. Inhibitors of the PI3-kinase/Akt pathway induce mitotic catastrophe in non-small cell lung cancer cells. Int. J. Cancer 119, 1028–1038. Hixon, M.L., Muro-Cacho, C., Wagner, M.W., Obejero-Paz, C., Millie, E., Fujio, Y., Kureishi, Y., Hassold, T., Walsh, K., Gualberto, A., 2000. Akt1/PKB upregulation

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